Method for screening for agents against cancer using an immunosuppression animal model

ABSTRACT

The invention relates to an animal model of cancer. The animal carries a tumour xenograft and is immunosuppressed by administration of cyclosporin and ketoconazole. The model is useful for studying cancer and treatment thereof.

[0001] This invention relates to a large animal model of human cancer,in particular in ruminant animals such as sheep which areimmunosuppressed by cyclosporin A and ketoconazole and which carrytransplanted human or murine tumours, or both. The invention alsorelates to the use of such an animal model in the study of cancer,particularly for evaluating candidates for radio-, chemo- orradiopharmaceutical therapy or radio-immunotherapy. The animal model isalso useful for radio-imaging of neoplasms or tumours, and for the studyof metastasis.

BACKGROUND OF THE INVENTION

[0002] At present there is no effective method available for treatmentof many solid tumours such as malignant melanoma or cancer of the colon,breast or ovary once the primary tumour has metastasised. Radiolabelledmonoclonal antibodies against tumour-associated antigens offer a uniquepotential for targeting radiotherapy to disseminated tumour cells whichmay ultimately lead to effective treatment of metastatic cancer.Radioimmunotherapy has been shown to be effective in haematologicalmalignancy, but problems of tumour localisation and penetration have sofar prevented successful treatment of solid tumour metastasis.

[0003] In order to evaluate therapeutic agents, or methods of imagingtumours, and to study the biological processes taking place in thedevelopment and metastasis of solid tumours, it is essential to useanimal models of cancer. The biodistribution of radiolabelled monoclonalantibodies can only be determined in the intact animal, where theinfluences of serum protein binding, vascular permeability, interstitialpressure and enzymatic breakdown all affect therapeutic radiation of thetarget tumour and determine the background irradiation of normaltissues. This essential dosimetry cannot be performed in vitro.

[0004] The immune-incompetent nude mouse, and less commonly, the nuderat, are the only models which are widely used for in vivo study ofhuman tumours. The tumours are usually transplanted subcutaneously inthese rodents. The major problem associated with human tumour xenograftsin nude animals is the disproportionate size of the tumour in relationto the total body weight of the animals, which precludes accurate,predictive pharmacokinetic studies of potential chemotherapeutic andradiopharmaceutical treatments for human cancer, and adversely affectsthe usefulness of such models for imaging studies.

[0005] Similar problems are encountered in orthotopic implantationmodels, in which human tumours or tumour cells are transplanted orinjected into the organ or tissue of origin in recipient immunodeficientathymic mice (Manzotti et al, 1993). Although metastasis of thetransplanted tumour is achieved, accurate and reliable data onusefulness of therapeutic agents or methods are still limited by thedisproportionate size of the tumour in relation to the total body weightof the mouse.

[0006] Therefore, a large animal model would be more suitable as a modelof cancer and for detailed study of targeted cancer therapy. Largeanimal models of human cancer are not readily available, because of thedifficulty of establishing tumours in such hosts; the xenografts usuallydo not grow or are rejected.

[0007] An animal model which would allow investigation of tumour nodulesof a specific size and location, and which would simulate patterns ofmetastasis in various types of cancer, is particularly desirable. Largeranimal models will also permit more effective and accurate evaluation ofpotential methods of therapy and imaging, and better characterisation ofthe biological events taking place during development and treatment ofsuch cancers.

[0008] One way of inducing acceptance of xenografts is theadministration of Cyclosporin A (CSA), a cyclic fungal peptide producedby Tolypocladium inflatum Gams. CsA is a neutral cycloundecapeptide withpotent immunosuppressive properties (Borel, 1989; Di Padova, 1989; Hesset al, 1988). This antifungal metabolite appears to inhibit both humoraland cellular immune responses by selectively interfering with T-cellactivation (Borel, 1989; Di Padova, 1989; Hess et al, 1988). CsA hasbeen shown to be effective in preventing transplant rejection in bothhumans and animals, but its use is often limited by its toxicside-effects (Borel, 1989; Reynolds et al, 1992; Russ, 1992), and by thehigh concentrations required in order to induce immunosuppression. Thenormal vehicle used, Cremaphor EL, can also induce severe toxic sideeffects.

[0009] For example, rabbits given intramuscular injections of CsA at 10mg/kg suffered from toxic side effects, and became anorexic anddeveloped pneumonia. These effects were only eliminated if largeranimals were used, and antibiotic and fluid therapy were institutedtogether with cyclosporin administration (Ligget et al, 1993). Cats alsorequire high oral doses of CsA in order to accept human tumourxenografts, since intravenous administration is also associated withspecies-specific Cremaphor-induced vasoconstriction with histaminerelease and anaphylaxis (Bowers et al, 1991).

[0010] However, in sheep, infusion of the castor-oil based vehicle forCsA, Cremaphor EL, is well tolerated (Tresham et al, 1988). There isalso no nephrotoxic reaction to intravenous CsA in sheep (Tresham et al,1990). A recent pharmacokinetic study of CsA administered intravenouslyto sheep revealed data similar to that reported in human transplantpatients (Charles et al, in press), and no toxic effects were described.

[0011] In addition to the toxic effects of CsA, a major disadvantage ofthis compound is the requirement for daily injections, which is bothtedious and expensive and limits the period of time within which animalscan be kept for observations (Hu et al, 1994, and de Ward-Siebinga etal, 1994). In all the studies mentioned above, the amount of CsAadministered has been more than 10 mg/kg of animal weight.

[0012] There has been a single brief report of experiments in whichhuman melanoma tumours have been subcutaneously grown in dogsimmune-suppressed by oral CsA (Wiseman et al, 1991). This method,however, also requires high doses of CsA due to its limitedbioavailability from oral administration. The intravenous route isprecluded by the anaphylactic reaction of dogs to the Cremaphor vehiclein which cyclosporin is dissolved (Bowers et al, 1991).

[0013] More recently, several groups have reported the use ofketoconazole in conjunction with CsA as a means of reducing the dose ofCsA required in transplant patients to maintain immunosuppression andprevent graft rejection (Gandhi et al, 1992; Butman et al, 1991; Firstet al, 1991; Wadhwa et al, 1987). Ketoconazole is a synthetic imidazoledioxolane used primarily for the treatment of superficial fungalinfections, chronic mucocutaneous candidiasis and genital candidiasis(Bodey, 1992; Breckenridge, 1992; Borelli et al, 1979). Ketoconazoleindirectly enhances the bioavailability of CsA by inhibiting the hepaticcytochrome P-450 mixed function oxidase system which is primarilyresponsible for CsA inactivation in vivo (Breckenridge, 1992; First etal, 1991; Wadhwa et al, 1987). Increased bioavailability reduces thedose of CsA required for therapeutic efficacy, which, in turn, decreasesthe toxicity associated with its use.

[0014] Ketoconazole, in addition to its synergism with CsA in theinduction and maintenance of immunosuppression, has been reported toexert anti-tumour activity against certain types of cancer (Eichenbergeret al, 1989a; Mahler and Denis, 1992). Ketoconazole also acts in synergywith anti-neoplastic drugs (vinblastine, etoposide) to inhibit thegrowth of human prostate carcinoma cells in vitro (Eichenberger et al,1989b).

[0015] Similarly, CsA has been shown to inhibit cell division of bothnormal and malignant cells in vivo and in vitro (Borel, 1989; Di Padova,1989; Barbera-Guillem et al, 1988; Kreis and Soricelli, 1979). Of thecell lines tested, human and murine T cell lymphomas and leukaemias werefound to be sensitive to CsA-induced growth inhibition at doses of 0.5-5μg/ml, whereas non-lymphoid cell lines and certain murine B and nullcell leukaemias were insensitive to doses of up to 10 μg/ml (Borel,1989).

[0016] We have surprisingly found that concomitant oral administrationof ketoconazole and CsA to a mammal produces immunosuppression whichallows xenografting of cancer cells or tissues and provides a largeanimal model for the study of cancer. This is particularly unexpected,in view of the anti-tumour effects of ketoconazole and CsA, and thedifficulty of inducing and maintaining immunosuppression with non-toxicdoses of CsA.

[0017] A main advantage of the animal model according to the presentinvention is the cost effectiveness of obtaining and maintaining theanimals. No aseptic or sterile conditions are necessary and the animalscan be maintained on a normal diet.

[0018] Our model also permits investigation of tumour nodules of desiredsize at predetermined sites, which simulate the usual patterns ofmetastasis of particular cancers.

SUMMARY OF THE INVENTION

[0019] Thus, in one aspect the invention provides an animal model ofcancer, comprising a mammal which is immunosuppressed by administrationof cyclosporin and ketoconazole, and which carries a tumour xenograft.

[0020] Preferably, the mammal is selected from the group consisting ofsheep, goats, cattle, pigs or the like. More preferably, the mammal is asheep. In a particularly preferred embodiment, the mammal has aplurality of xenografted tumours implanted subcutaneously.

[0021] Tumour cell lines which may be used in this model include but arenot limited to cells from solid tumours, such as those present in cancerof the colon, breast or ovary, or melanoma. Cell lines or spheroidsderived from cancerous cells are particularly useful for the purposes ofthe invention, for example LS174T, HT-29 and colon cancer and SKMELmelanoma cell lines. The tumour may be of human or non-human origin, butis preferably of human or murine origin.

[0022] It is particularly preferred that the tumours are introduced intothe model of the invention using orthotopic transplantation.

[0023] In a particularly preferred embodiment of the invention, tumourcells or tumours are transplanted into the host animal using Matrigel asthe vehicle. Matrigel is a reconstituted basement—-membrane preparationwhich facilitates tumour uptake at sites of incubation.

[0024] In a second aspect, the invention provides a method of evaluatingthe efficacy of a putative therapeutic agent against cancer, comprisingthe step of administering said agent to a ruminant mammal model of theinvention.

[0025] The agents which may be tested in this model include but are notlimited to immunochemotherapeutic agents, cytokines, chemotherapeuticagents and radiopharmaceuticals, and may also comprise internal orexternal radioactive agents as well as radiolabelled peptides. Genetherapy may also be evaluated using this model.

[0026] In a third aspect, the invention provides a method of evaluatingthe efficacy of a method of radioimaging of tumours or neoplasms,comprising the step of administering a radiolabelled, tumour-specificantibody to the ruminant mammal model of the invention.

[0027] The radiolabelled antibody may be a monoclonal or polyclonalantibody comprising a radiolabel, preferably selected from the groupconsisting of Technetium-99m, Indium-111, Iodine-131, Rhenium-186,Rhenium-188, Samarium-153, Lutetium-177, Copper-64, Scandium-47,Yttrium-90. Monoclonal antibodies labelled with therapeuticradionuclides such as Iodine-131, Rhenium-188, Holmium-166, Samarium-153and Scandium-47, which do not compromise the immunoreactivity ofantibodies and are not broken down in viva, are especially preferred.The person skilled in the art will appreciate that other radioactiveisotopes are known, and may be suitable for specific applications.Similarly it will be clearly understood that the term “antibody”encompasses fragments and analogues such as Fab, Fv and ScFv, providedthat the binding activity is retained. Peptide fragments of antibodiesare specifically contemplated by the invention. The fragments oranalogues may be prepared using recombinant DNA methods or by syntheticmethods such as solid-phase synthesis. The radioimaging may be conductedusing Single Photon Emission Computer Tomography (SPECT), PositionEmmission Tomography (PET), Computer Tomography (CT) or MagneticResonance Imaging (MRI). Correlative imaging, which permits greateranatomical definition of location of metastases located byradioimmunoimaging, is also contemplated.

[0028] In a fourth aspect, the invention provides a method of screeningof therapeutic radiolabelled peptides directed against tumours,preferably tumour-associated receptors, antigens or ligands or the like.Therapeutic radiolabelled peptides such as 90 Yttrium-labelledoctreotide or ¹¹¹Indium-labelled octreotide are contemplated.Radiolabelled antibodies to tumour-associated ligands or antigens andtherapeutic agents linked to such entities are also within the scope ofthe invention.

[0029] In a fifth aspect, the invention provides a method of producing aruminant mammal bearing a tumour xenograft, comprising the step ofconcomitant administration of CsA and ketoconazole to the mammal.Preferably the ketoconazole is administered in a drench formulationwhich by-passes the rumen and is absorbed in the abomasum. This provideshighly reproducible bioavalability and predictable competitiveinhibition of CsA metabolism in the liver.

[0030] Preferably, the dose of CsA is in the range 2.5 to 3.5 mg per kgadministered twice a day and the dose of ketoconazole is 5 to 10 mg perkg administered twice a day. In a particularly preferred embodiment, 10mg/kg ketoconazole is administered twice a day to maintain trough serumlevels of CsA within the range 1000-1500 ng/ml.

[0031] The model system of the invention enables the testing oftherapeutic methods directed against primary malignancy or metastaticcancer in a manner which has hitherto not been possible. The model issuitable for testing of radiotherapy, immunotherapy (including the useof cytokines), chemotherapy, and gene therapy. The model is also usefulfor testing of targeting or localisation agents, methods of imaging, andmethods for monitoring the progress of therapy.

[0032] In a sixth aspect, the invention provides a method of directtransplantation of a xenogeneic tumour, comprising the step oftransplanting a surgically-removed specimen into a mammal which isimmunosuppressed by administration of cyclosporin and ketoconazole andallowing the specimen to metastasize in said mammal.

[0033] In a seventh aspect, the invention provides a method-ofstimulating spontaneous metastasis of tumour cells to a target site suchas the liver or lymph nodes, comprising the step of transplanting saidcells to a mammal which is immunosuppressed by administration ofcyclosporin and ketoconazole and allowing the cells to metatasize insaid mammal.

[0034] In a eighth aspect, the invention provides a compositioncomprising a CsA or cyclosporin-like compounds and ketoconazole orrelated compounds, together with a pharmaceutically acceptable carrier.

[0035] In a ninth aspect, the invention provides a kit comprising a CsAor cyclosporin-like compound and ketoconazole or a related compound,wherein the ketoconazole or related compound increases thebioavailability of the CsA or cyclosporin-like compound and enhances theestablishment of tumour xenografts.

[0036] It will be clearly understood that, although the invention hasbeen described in detail with reference to immunosuppression using CsAwhose bioavailability is enhanced with ketoconazole, the invention alsocontemplates the use of immunosuppressive compounds related to CsA, suchas those disclosed in U.S. Pat. No. 4,117,118, synthetic or naturalanalogues of CsA such as CsB to I, or the compounds disclosed inAustralian Patent No. 660623 by Vertex Pharmaceuticals, Inc.

[0037] In addition, there are other compounds which the person skilledin the art will recognise as being suitable to improve bioavailabilityof CsA, such as compounds related to ketoconazole (including, but notlimited to, fluconazole), and calcium channel blockers.

[0038] Without wishing to be bound by any proposed mechanism for theobserved advantages, it is believed that ketoconazole, which bears nochemical structural relationship to CsA, competes with hepatic enzymeswhich break down CsA.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The invention will now be described by way of reference only tothe following non-limiting examples, and to the figures, in which:

[0040]FIG. 1 is a correlation plot for CSA concentration estimated in136 blood samples by enzyme multiplied imnunoassay (EMIT®) and by highperformance liquid chromatography (hplc). The solid line shows the lineof best fit as calculated by linear regression.

[0041]FIG. 2 shows the relationship between the difference between CsAconcentration estimated by EMIT and hplc (y-axis) versus the mean of theconcentrations measured by EMIT and hplc (x-axis).

[0042]FIG. 3 shows the pharmacokinetic concentration-time profiles forCsA (3 mg/kg intravenously (iv) in sheep #14 in Example 1, after thefirst dose ; day 0) and at steady-state (; day 18) in the presence ofketoconazole (10 mg/kg orally (po). The solid lines show the log-linearregression fitted line for the last 5 data points.

[0043]FIG. 4 is the area under the blood concentration—time curve, AUC,over 24 hours following CsA administration. The dose of ketoconazole was10 mg/kg. The full dose of CsA was 5 mg/kg and the half dose was 2.5mg/kg. Asterisks indicate significantly different AUC values relative toa full dose of CsA alone administered iv (p<0.05, Dunnett's test).

[0044] FIGS. 5(a), (b) and (c) show the mitogen-stimulated lymphocyteproliferation responses after CsA (5 mg/kg iv) administration.Individual and mean responses of 5 individuals to ConA (a), PHA (b) andPWM (c) administration are shown as a percentage of the mean change incounts per minute (Δ cpm) of 5 sheep. Interassay coefficients ofvariation (based on background cpm data from individual sheep assayed on6 different days) were between 75.7-126.6%.

[0045] FIGS. 5(d), (e) and (f) show the mitogen-stimulated lymphocyteproliferation responses after CsA (5 mg/kg iv) administration withketoconazole (10 mg/kg). Individual and mean responses of 6 individualsto ConA (d), PHA (e) and PWM (f) are shown as a percentage of the meanday 0 delta cpm of 6 sheep. Interassay coefficients of variation (basedon background cpm data from individual sheep assayed on 6 differentdays) were between 39-161%.

[0046]FIG. 6 shows the ED₅₀ of CsA and ketoconazole in three tumour celllines—B16M, HT-29 and SKMEL, represented as tumour cell growth (OD 590nm) in the presence of increasing concentrations of CsA (0-60 μg/ml) andketoconazole (0-60 μg/ml) (log scale). A representative CsA andketoconazole growth inhibition curve is shown for each cell line.

[0047]FIG. 7 shows the isobologram analysis of the effects ofcombination CsA/ketoconazole on tumour cell growth in vitro. The ED₅₀sof CsA in the presence of 3 sub-optimal concentrations of ketoconazole(no circle) and ketoconazole in the presence of 3 sub-optimalconcentrations of CsA (circled) are presented as the FIC for eachreagent. A representative isobologram is given for each cell line. Thedotted line depicts the expected shape of the curve if the interactionis additive and is given for comparison.

[0048] FIGS. 8-15 show the effects of transplantation of various celllines into sheep immunosuppressed with cyclosporin and ketoconazole.Cells were inoculated as suspensions or spheroids with or withoutMatrigel, and tumour xenografts were examined macroscopically and fixedin formalin:

[0049]FIG. 8 is a photograph of SK-melanoma tumour deposit in skin.

[0050] Panel A shows diffuse sheets of malignant cells of “epithelioid”type with abundant amphophilic cytoplasm, vesicular nuclei and variablyprominent nucleoli. Moderate nuclear pleomorphism is seen and mitoticfigures are easily found. There is no evidence of necrosis and a minorchronic inflammatory cellular exudate is present at the periphery withno significant numbers of tumour infiltrating lymphocytes (H&E stain).

[0051] Panel B shows a positive granular cytoplasmic staining togetherwith some nuclear staining with the immunoperoxidase preparation S100.

[0052] Panel C is a Mason Fontana preparation which shows no definitepigment deposition.

[0053]FIG. 9 is a photograph of SK-melanoma deposit in lymph node.

[0054] Panel A illustrates sections of the lymph node and show extensivereplacement of the parenchyma by diffuse sheets of malignant melanomacells.

[0055] Panel B shows variable mild to moderate predominantly cytoplasmicstaining of tumour cells using the S100 preparation.

[0056]FIG. 10(i) is a photograph of skin deposit of adenocarcinomaLS174T.

[0057] Panel A shows well formed ancinar structures lined by tallcolumnar cells with moderate nuclear pleomorphism in the tumour. Mitoticfigures are easily identified and there is mild focal necrosis with nosignificant inflammatory cellular exudate. Tumour infiltratinglymphocytes are infrequent (H&E).

[0058] Panel B represents abundant intraluminal PAS positive diastaseresistant neutral mucin as seen with the PAS-D preparation.

[0059] Panel C shows immunostaining with Carcinoembryonic antigen.Marked positive, predominantly luminal staining with a lesser degree ofgranular cytoplasmic staining can be seen.

[0060]FIG. 10(ii) is a photograph of HT 29 adenocarcinoma deposit inskin.

[0061] Panel D shows that the tumour is poorly differentiated withlittle tendency towards acinar formation and is formed by predominantlysheet like arrangements of columnar and cuboidal cells with vesicularnuclei and prominent nucleoli. Mitotic figures are easily identified andthere is focal necrosis and some peritumoural fibrosis. No significantinflammatory cellular response or tumour infiltrating lymphocytes areseen.

[0062] Panel E shows that a small amount of intraluminal PAS positivepost diastase neutral mucin is seen in occasional acinar structurespresent. There are also intracytoplasmic mucin deposits, although thisis not marked.

[0063] Panel F represents the immunoperoxidase preparation for CEAantigen which shows mild variable granular cytoplasmic staining withoccasional “dot” like intracytoplasmic deposits.

[0064] The LS174T adenocarcinoma is well differentiated with prominentacinar formation, and exhibits marked mucinogenesis and markedpositivity with CRA. In contrast, HT29 adenocarcinoma is poorlydifferentiated with only a mild degree of mucinogenesis and mildvariable staining with CEA.

[0065]FIG. 11 is a photograph of LS174T adenocarcinoma deposit inintestinal wall.

[0066] There are deposits of moderate to well differentiatedadenocarcinoma present beneath the mucosa showing some peritumouralfibrosis and a mild chronic inflammatory host response but nosignificant necrosis (H&E stain).

[0067]FIG. 12 is a photograph of LS174T adenocarcinoma deposit in theliver.

[0068] There are deposits of moderately differentiated adenocarcinomapresent associated with a mild to moderate degree of peritumouralfibrosis and a moderate chronic inflammatory cellular exudate is alsoseen. However, no significant necrosis or tumour infiltratinglymphocytes are present.

[0069]FIG. 13 is a photograph of LS174T adenocarcinoma deposit inperitoneal wall.

[0070] Panel A shows a tumour deposit with a well differentiatedadenocarcinoma having prominent acinar formations lined by tall columnarcells with moderate nuclear pleomorphism and easily identifiable atypical mitoses. No significant necrosis is present and there is a mildperitumoural chronic inflammatory cellular exudate but no significantnumbers of tumour infiltrating lymphocytes are seen (H&E stain).

[0071] Panel B shows abundant intraluminal PAS positive diastaseresistant neutral mucin, seen with the PASD preparation.

[0072] Panel C shows immunostaining with Carcinoembryonic antigen,leading to marked positive, predominantly luminal staining as well asgranular cytoplasmic staining.

[0073]FIG. 14 is a photograph of JAM ovarian carcinoma deposit in ovary.

[0074] There are predominantly diffuse sheet like arrangements ofpleomorphic malignant cells showing variable cytologic features. Tumourgiant cells are prominent and there is moderate to marked nuclearpleomorphism with prominent a typical mitoses. No definite papillarystructures are present and there is a mild peritumoural inflammatoryhost response and no significant tumour necrosis present.

[0075]FIG. 15 is a photograph of JAM ovarian carcinoma in peritonealwall. before dilution to 50 mg/ml with sterile saline. Before oraladministration, crushed ketoconazole tablets were suspended in a drenchvehicle which contained silicon dioxide (Ultrasil VN3 colloidal silicondioxide, Degussa Australia Pty Ltd., Melbourne, Australia) gum xantham(Sigma), and polyethylene glycol 6000 (Sigma) in a citrate buffer.

[0076] Drugs

[0077] Acetonitrile was of hplc reagent grade. Fluoxetine hydrochloridewas obtained from Eli Lilly Australia Pty, Ltd (West Ryde, NSW), CsAfrom Sandoz Australia (North Ryde, NSW), and ketoconazole from JanssenCilag. All other chemicals were of analytical reagent grade.

[0078] Statistics

[0079] Data were analysed using the two-tailed Student's t test, unlessotherwise stated.

EXAMPLE 1 Pharmacokinetics of CsA in Sheep; Effects of Coadministrationof Ketoconzole

[0080] Surgical Procedure

[0081] Silastic catheters were placed to a depth of approximately 20 cmin both external jugular veins of Merino-Cross Dorset ewes under localanaesthesia, two days before drug administration commenced. Catheterswere taped/sewn to the lateral surface of the neck, sealed with a threeway tap, and protected with an elastic net bandage around the neck. Onecatheter was used for administration of CsA and the other for venousblood sampling. Catheters were flushed with sterile, heparinised salinetwice daily to maintain patency.

[0082] There are diffuse sheets of poorly differentiated tumour similarto that described above, associated with a mild to moderate peritumouralchronic inflammatory cellular exudate and some fibrosis. No significantnecrosis is seen and only small numbers of tumour infiltratinglymphocytes are present.

[0083] General Methods

[0084] Animals

[0085] Sheep

[0086] Mature Suffolk cross and Merino wethers were penned individuallybut at 4 to a room and were allowed to acclimatise for 10 days in ananimal holding facility. The ad libitum diet comprised sheep cubes (GlenForrest Stock Foods, WA.) supplemented by rough-cut chaff, hay andwater. These animals were used for studying the pharmacodynamics andimmunological effects of CsA and ketoconazole, and for implantation ofcell lines.

[0087] Lambs

[0088] Merino-Dorset cross-lambs-of approximately 12 weeks of age,weighing around 25 kg and bred at Murdoch University, Murdoch, WesternAustralia, were housed in the same manner as the sheep.

[0089] Reagents

[0090] Cyclosporin A (CsA) was kindly donated by Sandoz Pharma Pty Ltd(Basel, Switzerland). The powder was dissolved in ethanol 1.75% v/v andpolyethoxylated castor oil cremophor (BASF Chemicals, Melbourne,Australia) 3.25% v/v, then diluted to volume with sterile saline justprior to administration.

[0091] Ketoconazole was supplied as tablets (Nizoral, Janssen-Cilag,Lane Cove, NSW). Prior to administration by the intraperitoneal route,ketoconazole tablets were crushed and dissolved in a minimal volume ofmethanol Experimental Design and Blood Sample Collection Schedule

[0092] CsA pharmacokinetics were studied after the first dose (3 mg/kgiv) and again at steady-state when the animals had been receiving CsA (3mg/kg iv) and ketoconazole (10 mg/kg po) for 18 days. Following thefirst dose on the first study day, venous blood samples (5 ml,anticoagulated with EDTA) were collected at 0.17, 0.33, 0.5, 0.75, 1, 2,3, 4, 6, 8, 12, 15, 22, 27 and 32 h. Following this initial study, twicedaily administration (dose interval τ=12 h) of CSA and ketoconazole wascommenced. Blood samples for CsA analysis were obtained twice weekly,just before the morning doses, over the next 18 days. Animals wereweighed twice weekly and absolute drug doses were modified to maintainthe initial dose rates in mg/kg. on day 18, when the CsA bloodconcentrations had reached steady-state, the final CsA dose wasadministered and blood samples were again collected at 0.17, 0.33, 0.5,0.75, 1, 2, 3, 4, 6, 8, 12, 15, 24, 30, 36, 48, 56, 72, 96 and 144 h.Ketoconazole administration was continued throughout the latter bloodsampling period.

[0093] Analysis of CsA and Ketoconazole

[0094] For routine monitoring of blood CsA concentrations during theexperimental period, CsA in whole blood was measured by enzymemultiplied immunoassay (EMIT®, Syva Company, Evergreen, Calif.) aspreviously described (Dusci et al, 1992). Blood samples were then frozenat −20° C. and at the end of the experiment were analysed by highperformance liquid chromatography (hplc) as previously described (Dusciet al, supra). Only hplc-derived data were used for subsequentpharmacokinetic analyses.

[0095] Some blood samples taken on days 18-23 were centrifuged to yieldplasma which was frozen at −20° C. until analysed for ketoconazole by aspecific hplc method. Aliquots of plasma (0.1 ml) were vortexedvigorously with 1 ml acetonitrile (containing 8 mg of fluoxetinehydrochloride as internal standard) for 15 sec. The mixture wascentrifuged at 1200 g for 10 min to sediment proteins, 0.5 ml of thesupernatant was aspirated, transferred to a clean tube and evaporated todryness at 50° C. using dry N₂. The extract was reconstituted in 0.2 mlof hplc mobile phase and 0.02 ml aliquots were injected onto the hplccolumn. The hplc system consisted of a Merck RP Select B C18 column (25cm×4.6 mm id), a mobile phase of 55% acetonitrile in 0.01% v/v H₃PO₄ and0.01% W/V NaCl. The solvent was pumped at a flow rate of 1.5 ml/min andeluting compounds were detected by their UV absorbence at 210 nm. Theassay was linear over the range 0.5-33 mg/l with a detection limit of0.2 mg/l. The within-day coefficients of variation at 2, 5.6 and 22 mg/lwere 3.6, 6.8 and 3.0% respectively (n=5). The between-run coefficientof variation at 2 mg/l was 9.8%(n=5).

[0096] Pharmacokinetic Analyses

[0097] Whole blood concentration-time data for cyclosporin were analysedby a noncompartmental pharmacokinetic method using the program TOPFIT(Thomann, 1993). The terminal elimination rate constant (λ_(z)) wasestimated by log-linear least squares regression of the last 6-8concentration-time data points. Areas under the plasma-concentrationtime data and (AUC₀₁₂ at steady-state and/or AUC_(0-∞)) were measured bythe linear trapezoidal rule with area from the last measuredconcentration to infinity being estimated as C_(p last/Ke). Plasmaclearance (CL=dose/AUC), mean residence time (MRT=AUMC/AUC andMRT_(ss)={AUMC_(0-τ))+τAUC_((τ-∞))}/AUC_((0-τ))), volume of distribution(V_(z)=CL/λ_(z)), and volume of distribution at steady-state(V_(ss)=MRT*CL) were estimated from the whole blood data as appropriate.Results have been summarised as mean (95% confidence interval).Differences between means were assessed by a paired t test, at the 0.05level of significance.

[0098] Results

[0099] Correlation Between EMIT and hplc Methods of Analysis

[0100]FIG. 1 shows that there was a significant correlation(EMIT=0.8981×hplc+110.7; r²=0.989) between CsA concentrations in 136blood samples measured by the EMIT immunoassay system and by thespecific gold standard hplc method. However, when the data arecritically analysed using the plot of the difference between the twomethods versus the average of the EMIT and hplc methods (FIG. 2; Blandand Altman, 1986), it is apparent that the EMIT method performssatisfactorily up to around 2000 μg/l, but at greater concentrations, itconsistently underestimates the true concentration. The reason for thisdiscrepancy has not been identified, but differences in the ratio of CsAto its metabolites between low (mainly from the first study on day 0)and high (mainly at steady-state with ketoconazole present) CsAconcentrations may be a modulating factor in the specificity of the testkit antibodies. While the nature of the difference between these twomethods for CsA assay in sheep blood is different to that which we havepreviously reported for human blood (Dusci et al, 1992), the dataindicate that the specificity of immunoassay methods is oftenquestionable. Thus, only concentration measurements made by a specifichplc method are satisfactory for pharmacokinetic analyses. Nevertheless,we consider that the EMIT method is adequate for rapid routinemonitoring of trough concentrations of CsA in the immuno-suppressedsheep model, particularly as we have found that these concentrationsshould be maintained in the range of 750-1500 ng/l.

[0101] Steady-State Concentrations of Ketoconazole

[0102] Steady-state concentrations of ketoconazole were measured inplasma from 5-8 trough blood samples obtained from the sheep on days18-23 of the study. Mean concentrations were 2.6, 2.19, 1.63 and 3.04mg/l in sheep 14 through 17 respectively.

[0103] Pharmacokinetics and Steady-state Concentrations of CsA Beforeand During Retoconazole Coadministration

[0104] Following regular dosing with CsA and ketoconazole twice daily,trough blood CsA concentrations increased slowly and plateaued afterabout 2 weeks. Mean trough concentrations for the 4 sheep were 925,1163, 954, 1694 and 1906 μg/l on days 3, 8, 10, 13 and 18 of treatment.Typical plasma concentration-time profiles for sheep #14 after the firstdose and at steady-state are shown in FIG. 3. Mean pharmacokinetic datafor all 4 animals are summarised in Table 1. At steady-state, half-lifeand MRT were significantly increased (P<0.007) and CL was significantlydecreased (P<0.007) compared to the values for the first dose. BothV_(z) and V_(ss) were similar after the first dose and at steady-statein the presence of ketoconazole. TABLE 1 Pharmacokinetic descriptors forcyclosporin (3 mg/kg iv) after the first dose alone, and at steady-stateduring the coadministration of ketoconazole (10 mg/kg po). CL (ml/ CsAt_(1/2) (h) MRT (h) min/kg) V_(z) (l/kg) V_(SS) (l/kg) First  14.7  12.6 9.47 11.5  6.9 dose  (5.1-24.3)  (4.2-21.0) (6.2-12.7) (5.5-17.5)(2.8-11.0) Stead-  72.0*  75.4*  1.62* 10.1  7.3 y-state (38.6-105.4)(41.8-109.0) (1.38- (5.5-16.3) (4.1-10.5)  1.86)

[0105] Summary

[0106] The inhibition of CSA metabolism by other drugs was first notedthrough drug interaction studies (Yee, 1990). More recently, there havebeen several clinical studies which have shown that coadministration ofeither the competitive inhibitor ketoconazole (First et al, 1993; Pattonet al, 1994; Keogh et al, 1995) or the competitive substrate diltiazem(Smith et al, 1994; Valantine et al, 1992; Wahlberg et al, 1992) has asubstantial dose-sparing action on CsA in transplant patients.

[0107] Our values for MRT, CL and V_(ss) for CsA following the firstdose were comparable to those reported by Charles et al (1993). Ourstudy is the first to show that coadministration of ketoconazolesignificantly decreases the CL of CsA in the sheep. MRT wassignificantly reduced (approximately 6-fold), V_(ss) was unchanged, andthere was a corresponding significant decrease in CL. These changes areconsistent with data for the iv use of CsA in humans (Gomez et al,1995). Thus, we conclude that ketoconazole can be used successfully as aCsA sparing agent in the sheep model.

EXAMPLE 2 In vivo/In vitro Assessments of Single Dose Pharmacodynamicsand Immunological Effects of CsA and Ketoconazole in Sheep.

[0108] Treatment Procedure

[0109] On the day on which drug treatment was initiated, each sheep wasfitted with an intrajugular cannula to facilitate the taking of bloodsamples. Cannulae remained in situ for 24 to 48 hours only and weremaintained with a solution containing heparin, penicillin andstreptomycin and were flushed with sterile saline prior to taking bloodsamples for pharmacokinetic studies. CsA was given by injection into theopposite external jugular vein and subsequent blood samples forlymphocyte culture were obtained by venipuncture from the oppositeexternal jugular vein. In pharmacokinetic studies, blood was sampled inEDTA collection tubes immediately before administration of CsA and atintervals of 20 min, 30 min, 45 min, 60 min, 1.5 hours, 2 hours, 3hours, 6 hours, 9 hours, 12 hours, 24 hours, 36 hours and 48 hours afterreceiving CsA. Heparinized blood samples for lymphocyte function assayswere collected before administration of CsA (Day 0) and at 24 hours, 48hours, 72 hours, 5 days and 7 days after receiving CsA. Whereketoconazole was administered in combination with CsA, intraperitoneal(ip) ketoconazole (5 mg/kg) was commenced one day prior to CsA, or oralketoconazole (10 mg/kg) was commenced two days prior to CsA, to allowfor an effect on the liver before exposure to CsA.

[0110] CsA Assay

[0111] Levels of CsA in peripheral blood were measured using the CsAMonoclonal Whole Blood fluorescence polarization immunoassay system(Abbott Diagnostics, Abbott Park, Ill., USA) and read on a TdXautoanalyser.

[0112] Lymphocyte Preparation

[0113] Lymphocytes were prepared from heparinized whole blood collectedbetween 8.30 and 10 am on each test day. The leucocyte-rich buffy coatlayer was collected after separation, diluted with phosphate bufferedsaline (PBS), and applied to Ficoll-hypaque (Pharmacia, North Ryde, NSW)density gradients to obtain lymphocyte preparations. After two low-speedwashes with PBS, the lymphocyte preparations were resuspended in RPMI1640 medium (Flow Laboratories, Australia Biosearch, Karrinyup,Australia) supplemented with 10% foetal calf serum (FCS, Cytosystems,Sydney, Australia) and penicillin (100 μ/ml) before counting andconfirmation of viability.

[0114] Mitogen Stimulation Assays

[0115] The capacity of lymphocytes to proliferate in response to invitro stimulation by Phytohaemagglutinin A (PHA; Sigma), Concanavalin A(ConA; Sigma) and Pokeweed Mitogen (PWM; Sigma) was assessed.Lymphocytes were seeded into 96-well flat bottom tissue culture plates(Disposable Products, Adelaide, South Australia) at a concentration of10⁵ cells/well. Mitogens (PHA, ConA, and PWM) were added to triplicatewells to obtain concentrations of 10 μg/ml of each mitogen. Plates wereincubated at 37° C. in 5%CO₂ for 48 hours before labelling with 1MBq/well of ³H-thymidine (Amersham, Melbourne, Australia). Cultures wereharvested on to glass filters 24 hours after labelling using anautomated cell harvester (PHD, Cambridge Technology, USA) and ³Hincorporation determined by liquid scintillation counting using a Minaxitri-Carb 400 beta counter (United Technology/Packard). Results wereexpressed as change in counts per minute (Δcpm), constituting the meancpm of triplicate stimulated wells minus the mean cpm of triplicatebackground wells without added mitogen.

[0116] Mixed Lymphocyte Cultures

[0117] The ability of isolated lymphocytes to respond to allogeneictissue antigen was assessed by mixed lymphocyte culture (MLC).Lymphocytes obtained as described above from test sheep were adjusted to2.5×10⁵/well in 96-well flat bottom tissue culture plates. An equalnumber of lymphocytes isolated from an unrelated untreated donor sheepwere subject to 20 Gray (Gy) of X-irradiation and added to triplicate orquadruplicate wells as stimulator cells. Irradiated lymphocytes fromeach individual test sheep were also prepared and set up againsthomologous responder cells to obtain individual background data.Controls to confirm adequacy of inactivation by X-irradiation were alsoincluded in each assay.

[0118] Mixed lymphocyte cultures were incubated at 37° C. and 5% CO₂ for6 days before overnight labelling with ³H-thymidine and harvesting asdescribed above. Stimulation indices were determined by dividing themean cpm of the wells containing test lymphocytes co-cultured withX-irradiated stimulator cells by the mean cpm of the background wellsutilizing X-irradiated and non-irradiated homologous cells.

[0119] FACS Analysis

[0120] Lymphocytes prepared as described above were adjusted to 1 to3×10⁶/well in 96—well round bottom culture plates (Disposable Products,Adelaide, South Australia) and incubated overnight at 4° C. withmonoclonal antibodies to lymphocyte marker antigens at a final dilutionof 1/100. A panel of monoclonal antibodies defining ovine lymphocytesurface markers (Hein et al, 1991) was obtained from the Centre forAnimal Biotechnology, The University of Melbourne, Parkville, Australia.The monoclonal antibodies used were SBU-LCA (detecting CD45,leucocyte-common antigen), SBU-T1 (CD5, all T cells), SBU-T4 pool (CD4,T helper cell subset), SBU-T8 (CD8, T suppressor/cytotoxic subset) andSBU-T19 (defining the CD4-CD8-gamma delta T cell subset in sheep,currently of unknown function). B lymphocytes were identified by surfaceimmunoglobulin expression.

[0121] The following day, plates were washed three times with PBS andincubated with fluorescein-conjugated anti-mouse Ig (SilenusLaboratories, Hawthorn, Australia) at 1/100 dilution for 1 hour at 37°C. B cells were labelled by incubation with fluorescein-conjugateddonkey anti-sheep Ig (Silenus Laboratories, Hawthorn, Australia) at1/100 dilution. After a further three washes in PES, 50 μl of PBScontaining 1% formalin was added to each well and the plates stored inthe dark at 4° C. until FACS analysis was carried out using an EPICSProfile Analyser (Coulter Corporation, Hialeah, Ill., USA).

[0122] Biochemistry

[0123] Blood samples were taken daily over the course of the experimentsfor assessment of serum albumin, bilirubin, alkaline phosphatase, gammaglutamyltransferase (GGT), alanine aminotransferase (ALT), creatine,urea, and electrolytes to indicate liver and kidney function.

[0124] Statistics

[0125] Pharmacokinetic parameters were calculated by standard methods ofcurve fitting using the “MINIM” software package and a Macintoshcomputer. Area under the blood concentration-time curve (AUC) and thearea under the first moment blood concentration-time curve (AUMC), meanresidence time (MRT), steady state volume of distribution and totalclearance were calculated by standard methods (Gibaldi, 1991).

[0126] Results

[0127] Pharmacodynamics

[0128] The mean absorption time, half-life and steady state volumedistribution of CsA were comparable to those from humans (Gupta et al.,1987, Charles et al., 1993).

[0129] Ketoconazole significantly altered the AUC by reducing CsAclearance, leading to a two-fold increase in AUC after an oral dose ofketoconazole 10 mg/kg (p<0.05, Dunnett's test; 24,4 d.f.) the AUCfollowing a dose of 2.5 mg/kg of CsA with 10 mg/kg ketoconazole po wasslightly greater than that following 5 mg/kg CsA alone, as shown in FIG.4. Administration of ketoconazole either ip or po along with a halveddose (2.5 mg/kg) of CsA maintained the AUC at a level similar to thatachieved with the full dose of 5 mg/kg dose of CsA alone.

[0130] The pharmacokinetic parameters of CsA and ketoconazole,calculated using AUC and AUMC are shown in Table 2 and also FIG. 4.TABLE 2 Steady state Total clearance distribution volume AUC MRT Halflife Relative AUC* Treatment Group n 1/h/kg 1/kg ng/ml. h h h % CsA 5mg/kg IV 8 0.498 ± 0.024 5.719 ± 0.267  10183 ± 4.55 11.60 ± 0.63 8.04 ±0.44 CsA 5 mg/kg IV 6 0.358 ± 0.036 4.390 ± 0.467  14698 ± 1505 12.50 ±1.28 8.66 ± 0.88 144.3 plus Ketoconazole 5 mg/kg IP CsA 2.5 mg/kg IV 60.337 ± 0.045 2.447 ± 0.175  8049 ± 965  7.82 ± 0.97 5.42 ± 0.67 158.0plus Ketoconazole 5 mg/kg IP CsA 2.5 mg/kg IV 6 0.247 ± 0.012 3.490 ±0.238 10257 ± 536 14.08 ± 1.23 9.75 ± 0.85 201.0 plus Ketoconazole 10mg/kg PO

[0131] Mitogen Responses

[0132] Mitogen proliferation data from sheep receiving CsA 5 mg/kg aloneiv are shown in FIG. 5(a), (b) and (c). The data show that there was atransient suppression of lymphocyte response at 24 hour.

[0133] The concomitant ip administration of ketoconazole 5 mg/kg and CsAiv 5 mg/kg markedly potentiated the depression of lymphoproliferativeresponses to all mitogens tested (FIGS. 5(d), (e) and (f)). Normalreactivity to mitogens was regained at 48 hours in sheep, treated withCsA alone. In CsA+ketoconazole treated sheep responses substantiallyrecovered by 48 hours.

[0134] A correlation between CsA AUC values and depression of mitogenresponse was observed when comparing the CsA and the CsA+ketoconazoletreated groups (p<0.05)

[0135] MLC Responses

[0136] MLC responses were not significantly different from Day 0 at 48hours, 72 hours or 7 days, (p>0.05, Wilcoxon Signed Rank test, n=6).However lymphocyte responses were significantly elevated, relative toDay 0, at 5 days after receiving CsA (p<0.05, Wilcoxon Signed Rank test,n=6).

[0137] Lymphocyte Phenotypes

[0138] In sheep receiving CsA 5 mg/kg alone by the iv route numbers ofcirculating T1⁺ cells were slightly elevated at 48 hours (p<0.05,Student's t test, 2 d.f); the T4:T8 ratio remained unchanged.

[0139] In the experimental group receiving CsA 5 mg/kg i.v andketoconazole ip, the total number of T cells (T1+) did not altersignificantly but a significant increase in the number of circulating T4positive lymphocytes was observed. Likewise T4:T8 ratios were elevated.These results are summarized in Table 3. TABLE 3 Lymphocyte Subsetsafter Combined CsA and Ketoconazole Surface Antigen LCA T1 T4 T8 T119Slg  0 hrs 98.7 ± .92 62.3 ± 12.7  7.64 ± 1.76  4.9 ± 1.76 23.8 ± 7.219.6 ± 15.1 24 hrs 94.8 ± 6.6 54.1 ± 17.7 11.9 ± 2.4 9.0 ± 4.6 26.4 ±5.9 22.6 ± 14.4 48 hrs 87.9 ± 11.3 49.3 ± 8.2 13.7 ± 2.2 7.3 ± 2.6 26.4± 5.9 27.3 ± 11.7 72 hrs 92.0 ± 0.8 58.0 ± 9.2   13 ± 3.8 6.8 ± 2.1 20.4± 7.6 8.35 ± 6.6  5 days 97.3 ± 1.1 67.7 ± 6.9 11.85 ± 1.4  6.05 ± 1.9420.8 ± 4.6 12.9 ± 7.7  7 days 98.7 ± 2.6 63.3 ± 12.4 17.9 ± 3.1 6.6 ±1.4 18.98 ± 3.8  32.2 ± 11.1

SUMMARY

[0140] These results demonstrate that a single dose of CsA effectivelysuppressed lymphocyte responses in the sheep, and that the effect ofconcomitant ketoconazole administration on CsA kinetics in the sheepparallels that seen in humans.

[0141] Co-administration of CsA with ketoconazole was effective insuppressing T cell immunity in the sheep, and a single-dose regimen waswell tolerated, without apparent adverse reactions.

Example 3 Effects of CsA and Ketoconazole on Tumour Cell Growth in vitro

[0142] Prior to initiating tumour xenograft transplantation in a sheepmodel, the susceptibility of the tumour xenografts to the growthinhibitory effects of ketoconazole and CsA was determined. The tumourcell lines used in this study included a human colon carcinoma, HT-29, ahuman malignant melanoma, SKMEL, and a murine malignant melanoma, B16M.These tumour types were chosen because they represent commonmalignancies with high metastatic potential and high morbidity andmortality. In addition, relatively specific monoclonal antibodies(MoAbs) are available for all of these cell lines (DiMaggio et al,1990). The experiment was designed to evaluate the growth inhibitoryeffects of CsA and ketoconazole, used alone or in combination, on theHT-29, SKMEL and B16M tumour cell lines in vitro.

[0143] GENERAL

[0144] Tumour Cell Line

[0145] Tumour cell lines HT-29, SKMEL and B16M were obtained from theAmerican Type Culture Collection (ATCC, Rockville, Md., USA) andmaintained in RPMI-1640 medium, (Flow Laboratories, AustralianBiosearch, Karrinyup, Australia), containing 10% foetal calf serum (FCS,Cytosystems, Sydney, Australia), 2 mM L-glutamine, 100 mM sodiumpyruvate, 100 mM non-essential amino acids, (all from Aust. Biosearch),and benzylpenicillin (100,000 units/l, Commonwealth Serum Laboratories(CSL), Parkville, Australia). All tumour cell lines were incubated at37° C./5%CO₂/95% humidity and the medium was changed every 3 to 4 days.For passage and assay, cells were detached with 0.1% w/v trypsin/0.02%w/v versene (CSL).

[0146] MTT Assay

[0147] 3-(4,5-dimethylthiazo-2-YL)-2,5-diphenyl-tetrazolium bromide(MTT) was obtained from Sigma Corporation (St. Louis, Mo, USA). Theassay was performed according to the method of Mosmann (1983). Ingeneral, following incubation of the tumour cells with the CsA and/orketoconazole (see below), the plates were centrifuged (1000 g, 5 min)and 100 μl of the supernatant removed. 20 μl of MTT (stock at 5 mg/ml inPBS) was added to each well and the plates incubated at 37° C. for 4hours. 100 ul of 0.04N HCl-isopropanol was then added to each well andthe dark blue formazan-crystals were dissolved by mixing. Opticaldensities (ODs) were determined at 590 nm using a Titertek multiscanphotometer (Flow Labs.) and were directly proportional to cell growth(Mosmann, 1983).

[0148] In Vitro Assessment of Tumour Cell Growth

[0149] Tumour cells were seeded at 5 to 10×10³/well (100 μl/well),incubated overnight at 37° C., and exposed to CsA or ketoconazole (0-60μg/ml) for 1-3 days (final volume, 200 μl). Tumour cell growthinhibition was assessed using the MTT assay.

[0150] Results

[0151] The results are presented as the estimated dose of each reagentrequired to reduce tumour cell growth by 50% (ie the ED₅₀) as determinedfrom the linear portion of the growth inhibition curve, and are shown inFIG. 6 and Table 4. TABLE 4 ED₅₀ of CsA and Ketoconazole on B16M, HT-29and SKMEL in vitro.^(a) Test Reagent Tumor Cell Line I CsA B16M HT-29SKMEL days in culture: ED₅₀ (μg/ml) day 1 28.8 ± 3.8 29.6 ± 3.6 31.5 ±2.1 day 2 18.5 ± 1.0 23.3 ± 2.9 30.0 ± 1.2 day 3 10.2 ± 3.3 15.3 ± 3.225.5 ± 4.1 EtOH/Cremophor/saline vehicle: vehicle conc. (%/ml) day3-ED₅₀ (0.035%) (0.053%) (0.08%) II Ketoconazole B16M HT-29 SKMEL daysin culture: ED₅₀ (μg/ml) day 1 22.8 ± 2.8 23.8 ± 2.5 31.0 ± 2.6 day 221.2 ± 4.3 18.2 ± 1.8 30.0 ± 1.1 day 3 18.2 ± 3.9 12.7 ± 2.0 28.3 ± 1.7EtOH/Methanol vehicle: vehicle conc. (%/ml) day 3-ED₅₀ (0.06%)  (0.035%)(0.85%)

[0152] ED₅₀ Determination of CsA and Ketoconazole

[0153] All three tumour cell lines tested were found to be moderately tohighly resistant to the growth inhibitory effects of CsA andketoconazole, demonstrating ED₅₀s at or well above the maximumtherapeutic plasma concentration for both reagents, ie. 0.10 μg/ml(Borel, 1989; Reynolds et al, 1992; Eichenberger et al, 1989a; FIG. 6,Table 4). The murine melanoma cell line, B16M, was found to be the mostsusceptible of the three tumour cell lines to CsA, demonstrating an ED₅₀of 10.2±3.3 μg/ml compared to 15.3±3.2 μg/ml and 25.5±4.1 μg/ml for thehuman HT-29 and SKMEL tumour cell lines, respectively. Conversely,ketoconazole was found to be most inhibitory to HT-29 tumour cellgrowth, demonstrating an ED₅₀ of 12.7±2.0 μg/ml compared to 18.2±3.9μg/ml and 28.3+1.7 μg/ml for B16M and SKMEL, respectively (FIG. 6, Table4). Neither the CsA vehicle (Cremaphor EL/EtOH/saline) nor theketoconazole vehicle (EtOH/methanol) inhibited tumour cell growth atconcentrations present in the ED₅₀ doses of CsA or ketoconazole for the3 tumour cell lines. Slight inhibition (10—-15%) of B16M and HT-29 cellgrowth was observed following 3 days exposure to theCremphor/EtOH/Saline vehicle at concentrations of 0.1%, and thisinhibition has been corrected for in all experiments.

[0154] Isobologram Analysis of Interaction Potential of CsA andKetoconazole

[0155] The interaction potential of the combination of CsA andketoconazole on tumour cell growth in vivo was determined by isobologramanalysis (Czarniecki et al, 1984). The ED₅₀s of CsA (0-60 μg/ml) incombination with 3 sub-optimal ketoconazole concentrations (determinedfrom the ketoconazole ED₅₀ for each line), and ketoconazole (0-60 μl/ml)in combination with 3 sub-optimal CsA concentrations, were determinedand plotted as a fractional inhibitory concentration (FIC) of the ED₅₀of CsA, or of ketoconazole alone, which has a designated FIC of 1. Basedon this comparison, if the FIC values of the CsA/ketoconazolecombination form a convex curve, the interaction potential is consideredantagonistic. If the values fall in a straight line the effects areadditive, and if the points form a concave curve, the effects aresynergistic. Confirmation of the synergistic, additive or antagonisticnature of the CsA/ketoconazole interaction was determined using theadditivity model (Welander et al, 1985). The fractional survival of theindividual reagents is determined and the two values are multipliedtogether. The product of the two values becomes the expected cellsurvival, and is compared with the observed fractional survival when thetwo reagents are tested together. A synergistic response is defined byan observed fractional survival which is 0.5 of the expected survival,an additive response by an observed fractional survival of 0.5 to 1.5times the expected survival, and an antagonistic or subadditive responseby an observed survival which is 1.5 the expected survival.

[0156] The isobologram analysis suggested that the growth inhibitoryeffects of CsA and ketoconazole, when used in combination, were additiveto subadditive on the B16M and HT-29 tumour cell lines and additive tosynergistic on the SKMEL tumour cell line, as shown in FIG. 7. Analysisof the data using the additivity model demonstrated that the growthinhibitory effects of the combination CsA/ketoconazole were additive onall three tumour cell lines, as shown in Table 5. Some CsA/ketoconazolecombinations did approach synergism (ie. observed/expected fractionalsurvival of <0.5), but in all cases, one or both of the reagents waspresent at a concentration ≧10 μg/ml (ie greater than the maximumtherapeutic dose in humans). These results are summarised in Table 5.TABLE 5 Interaction Potential of CsA and Ketoconazole on B16M, HT-29 andSKMEL¹ Reagents: Reagents: Reagents: Exp. Obs. Exp. Obs. Exp. Obs. CSA +Keto (OD) CSA + Keto (OD) CSA + Keto (OD) (μg/ml) B16M Obs/Exp. (μg/ml)HT-29 Obs/Exp. (μg/ml) SKMEL Obs/Exp. 1.0 3.75 0.727 0.731 1.01 2.5 3.00.680 0.671 0.99 5.0 6.25 0.688 0.682 0.9 7.5 0.699 0.701 1.00 6.0 0.5630.539 0.96 12.5 0.558 0.594 1.06 11.25 0.670 0.720 1.07 9.0 0.235 0.3201.36 18.75 0.546 0.620 1.13 2.5 3.75 0.826 0.820 0.99 5.0 3.0 0.5500.517 0.94 10.0 6.25 0.688 0.604 0.88 7.5 0.766 0.736 0.96 6.0 0.4560.444 0.97 12.5 0.558 0.512 0.92 11.25 0.699 0.636 0.91 9.0 0.190 0.1880.99 18.75 0.546 0.559 1.02 5.0 3.75 0.740 0.715 0.97 10.0 3.0 0.3000.212 0.71 20.0 6.25 0.564 0.513 0.97 7.5 0.687 0.648 0.94 6.0 0.2480.142 0.57 12.5 0.428 0.447 1.00 11.25 0.661 0.658 1.00 9.0 0.104 0.0540.52 18.75 0.422 0.386 0.91 2.5 1.5 0.735 0.798 1.09 2.5 3.75 0.6790.603 0.89 5.0 3.75 0.640 0.734 1.15 3.75 0.702 0.880 1.25 7.5 0.5590.623 1.11 7.5 0.577 0.628 1.09 7.5 0.511 0.583 1.14 11.25 0.353 0.3521.00 11.25 0.491 0.453 0.92 5.0 1.5 0.724 0.789 1.09 5.0 3.75 0.6320.519 0.82 10.0 3.75 0.627 0.602 0.96 3.75 0.696 0.798 1.05 7.5 0.5200.499 0.95 7.5 0.559 0.596 1.07 7.5 0.504 0.544 1.08 11.25 0.328 0.2330.71 11.25 0.473 0.342 0.72 10.0 1.5 0.785 0.750 0.96 10.0 3.75 0.4270.377 0.88 20.0 3.75 0.582 0.518 0.89 3.75 0.759 0.751 0.99 7.5 0.3510.265 0.76 7.5 0.525 0.4.0 0.82 7.5 0.547 0.419 0.77 11.25 0.221 0.1190.54 11.25 0.447 0.283 0.63

SUMMARY

[0157] These findings suggest that the anti-tumour activities of CsA andketoconazole, alone and in combination, on the HT-29, SKMEL and B16Mtumour cell line are minimal at doses which can be achieved in vivo.Thus the regimen of CsA and ketoconazole used to induceimmunosuppression in the sheep model should not interfere with theestablishment of human/murine tumour xenografts in situ which will serveas targets for the assessment of radiolabelled MoAb imaging and therapy.

Example 4 Transplantation of Cell Lines into Sheep

[0158] Tumour Cell Lines

[0159] The following human tumour cell lines are grown in the CellBiology Research Unit at Fremantle Hospital. 1. LS174T (ATCC) Coloncarcinoma 2. HT 29 (ATCC) Colon carcinoma 3. OVCAR-N1H3 (ATCC) Ovariancarcinoma 4. CRL 1803 TT (ATCC) Medullary cell carcinoma thyroid 5. SKMEL (ATCC) Melanoma 6. HTB 3477 (Oncogen) Breast 7. JAM Serous cystadenocarcinoma of ovary 8. Control B16M Murine melanoma

[0160] Cells were maintained in media recommended by the American TypeCulture Collection (ATCC). Specifically, the cell lines were maintainedin 75 cm² tissue culture flasks (Costar, USA) in RPMI 1640 (LifeTechnologies, USA) supplemented with 10% fetal calf serum (FCS) and100U/ml penicillin (CSL, Australia) and for NIH:OVCAR-3, 10% extra FCSand human insulin NIH:OVCAR-3 adenocarcinoma of ovary (Actrapid, NovoNordisk, Denmark) at 10 μg/ml were added to the media. Confluent cellswere harvested using trypsin-versene (CSL, Australia) and counted, thenwashed twice with PBS to remove FCS and resuspended at a concentrationof 10⁸ cells/ml immediately prior to injection into sheep. All tumourcell lines were incubated at 37° C./5% C0₂/95% humidity and the mediumwas changed every 3-4 days. For passage and assay, cells were detachedwith 0.1% w/v trypsin/0.02% w/v versene.

[0161] For some experiments, tumours were passaged in nude mice and thentransplanted into the immunosuppressed sheep as small solid tumourpieces approximately 2 mm×2 mm diameter.

[0162] Following acclimatization, weighing and shearing of the sheep, ajugular venous catheter was inserted subcutaneously under localanaesthetic, exiting the skin at the back of the neck.

[0163] Twice daily intravenous administration of 3 mg/kg cyclosporin wasgiven via the indwelling jugular vein catheter each day for up to 70days. A drench oral administration of ketoconazole was also given on adaily basis according to standard Murdoch University veterinarytechniques in sheep. Parenteral administration of CsA in sheep andconcomitant oral ketoconazole had minimal side effects in a controlledenvironment.

[0164] Haematological, biochemical and CsA assays were performed on aweekly basis on blood obtained directly from the jugular vein.

[0165] Reagents

[0166] CsA in powder form was dissolved in alcohol and Cremaphor ELaccording to the protocol developed at Fremantle Hospital.

[0167] Solutions were prepared on a weekly basis and diluted withphysiological saline as required for daily aliquots to provide a dose ofCsA determined by trough level assay. The typical daily dose of CsA was6 mg/kg given in an indwelling jugular vein catheter in 2 divided dosesgiven in the morning and evening.

[0168] The cyclosporin may be administered by a continuous infusion pumpinto the jugular vein to acheive greater control of serum Cs levels andminimize expensive drug use.

[0169] Ketoconazole powder was prepared in a drench formulation asfollows:

[0170] 36 g of ketoconazole was gradually added to about 400 ml ofsolution containing the following: 1. Ultrasil 6.96 g, 2. Citric Acid2.78 g, 3. Sodium Citrate 5.92 g, 4. Keltrol 1.46 g, 5. PEG 6000 20.9 g,6. MYRJ 15.3 g, 7. Potassium Sorbate 1.05 g,

[0171] and to which 0.78 ml of concentrated 10M HCl and 0.84 ml of 40%formalin had been added.

[0172] The resulting mixture was stirred for 30 minutes after which 200ml of warm tap water was added. The mixture was again left to stir forat least one hour, after which the volume was made up to a final totalof 720 ml. After mixing well, the solution was aliquoted and stored at4° C. The final concentration of ketoconazole in this solution was 50mg/ml and the dose given to the sheep may be 10 mg/kg or 1 ml of thesolution per 5 kg sheep weight. Prior to giving the ketoconazole tosheep, the solution should be well mixed to distribute any sedimentedpowder although care should be taken to avoid frothing or aeration ofthe solution.

[0173] The basic ingredients of the drench were derived from thestandard preparation used for oral administration of medication toruminants at the School of Veterinary Studies at Murdoch University. Thedrench formulation of ketoconazole ensures abomasal delivery andeffectively bypasses the rumen following oral administration to thesheep, and thus rendering the ketoconazole readily bioavailable.

[0174] The daily oral dose of ketoconazole was 20 mg/kg in 2 divideddoses, given in the morning and evening.

[0175] CsA and Ketoconazole Assays

[0176] CsA assays were performed at the Western Australian Centre forPathology and Medical Research using the EMIT® Cyclosporin Assays (SyvaCo, San Jose, Calif., USA) on blood taken immediately before the morningdose. These trough CsA levels were maintained in the optimum range forimmunosuppression between 750 and 1500 ng/ml.

[0177] Trough blood level assays performed at the Western AustraliaCentre for Pathological and Medical Research showed that ketoconazoleplasma concentration was in the range 2.5-3.5 mg/L.

[0178] Immunological Assessments

[0179] a) Mitogen Responses

[0180] Peripheral blood lymphocytes were tested on a weekly basis forcapacity to respond to a series of mitogens. Lymphocytes were obtainedfrom heparinized blood drawn from the anterior brachial vein bycentrifugation and separation on a Ficoll/hypaque gradient. Afterwashing, lymphocytes were dispensed into microlitre trays at 5×10⁶cells/ml with an equal volume (100 μl) of RPMI containingphytohaemagglutinin, concanavalin A or pokeweed mitogen at 5, 10, and 20μg/ml, together with 2-mercapto-ethanol, foetal calf serum andpenicillin/streptomycin. Cells were cultured in an atmosphere of 5% C0₂for 72 hours and were pulsed with ³H-thymidine at 0.5 μCi/well over thefinal 16 hours of culture. After harvesting, radioactivity of cells wascounted in the scintillation counter and stimulation indices determined:${SI} = \frac{{{cpm}\quad {mitogen}\quad {stimulated}\quad {cells}} - {{cpm}\quad {background}}}{{{cpm}\quad {nonstimulated}\quad {cells}} - {{cpm}\quad {background}}}$

[0181] The functional capacity of lymphocytes was determined weekly.

[0182] b) Lymphocyte Phenotyping

[0183] To identify perturbations in number and proportion of B and Tcells during chronic CsA induced immunosuppression, the phenotype oflymphocytes, prepared as above, was determined by immunofluorescenceassay using a range of commercially available ovine monoclonal reagents.After labelling, suspensions were assayed by fluorescence-activated cellsorting. These assays provide an assessment of fluctuations in total Band T lymphocyte populations as well as T cell subjects. The resultswere expressed as percentages of total lymphocyte numbers.

[0184] c) Monitoring of Skin Allografts

[0185] Comparison of skin allografts with skin homografts on a dailybasis constitutes an in vivo measure of cell-mediated immunity.

[0186] Skin and Tumour Transplantation Procedures

[0187] Five days after the initiation of CsA and ketoconazoleadministration, the animals were premedicated with rompun and subjectedto general anaesthesia using halothane via an endotracheal tube.

[0188] Full thickness skin autograft and heterograft transplantationswere performed on the flanks following standard procedures.

[0189] Cell Injections

[0190] Injections were given via a 21G needle as 0.1 to 0.3 ml cells inPBS with or without 0.1 ml Matrigel (Collaborative Biomedical Products,USA). For co-injection of cells with Matrigel all cells, syringes andneedles were pre-chilled on ice, and the needle and syringe held in theinjection site until the Matrigel had gelled.

[0191] A minimum of two human tumours was inoculated subcutaneously ineach sheep under the bare skin of the inguinal region. At laparotomy,subcapsular and hepatic intraparenchymal inoculation of at least 2tumour cell types was performed (one acting as the non-specific controlfor the labelled monoclonal antibody). Inoculation of the peritoneumunder direct vision was also performed at the sites which were markedwith a suture for ready identification at subsequent laparoscopies.Breast cancer cells were inoculated subcutaneously in the thorax andinto chest wall and pleura to simulate common sites of local spread.

[0192] (i) Skin sites

[0193] 10⁷ LS174T, SK-MEL, HT-29, OVCAR-3 and JAM cells in PBS wereinjected subcutaneously 5 cm apart on the shaved sides and flanks of thesheep at 4 injection sites for each cell line. Up to three differentcell lines were injected into a individual sheep. For biodistributionstudies 8 sheep received 4 subcutaneous injections each of LS174T; HT-29and SK-MEL.

[0194] (ii) Intra-Abdominal Injections

[0195] The following procedures were carried out aseptically underhalothane general anaesthetic in the animal operating room. Atlaparatomy the cells were injected with or without Matrigel using a 1 mltuberculin syringe and a 21G needle. Non-absorbing sutures were placed 2cm from injection sites for subsequent location at laparascopy orautopsy. The abdominal incision was closed in layers and the sheepmonitored in the recovery room until post-operative recovery wascomplete.

[0196] (a) Ovarian and Peritoneal Wall Injections:

[0197] Three sheep received 2×10⁷ JAM cells +0.1 ml Matrigel in theopposite ovary and two peritoneal wall sites. Before closure of theperitoneum OVCAR-3 cells were injected into the peritoneal cavity of onesheep.

[0198] (b) Colon, liver and peritoneal wall injection:

[0199] Four sheep received 10⁷ LS174T cells+0.1 ml Matrigel injectedinto 4 sites along the colon wall and 2 sites in the liver andperitoneal wall as well as one injection each in the liver andperitoneal wall of 10⁷ LS174T cells without Matrigel.

[0200] (iii) Solid Tumour Transplantation

[0201] Solid tumour pieces were harvested from nude mice and cut into 2mm×2 mm cubes for transplantation into the sheep. Each transplantationsite corresponded to the location of predilection for each tumour typein human metastasis.

[0202] Monitoring Tumour Growth

[0203] Skin tumours were measured weekly in two dimensions with calipersexcised from the skin at varying times and fixed in formalin forsubsequent histological examination. At autopsy 3-6 weeks after tumourcell inoculation appropriate organs and draining lymph nodes wereremoved and examined macroscopically and fixed in formalin forhistology.

[0204] Tissues were also fixed in neutral buffered formalin andprocessed through alcohol and xylene to paraffin.

[0205] Sections were cut at 4 μ on a rotary microtome, dried at 60° C.and stained with Harris haematoxylin (H&E stain) and aqueous eosin on arandom access staining machine. These were then coverslipped using aresin mounting medium and viewed under light microscopy.

[0206] Mucins in tissues were demonstrated using the periodicacid-Schiff reaction (P.A.S). Mucins were oxidised by periodate toexpose aldehydes which were demonstrated with Schiffs reagent. Anyglycogen in the tissue was removed by prior treatment with fresh maltdiastase.

[0207] Antigen demonstration was achieved by a peroxidase conjugatedstreptavidin staining procedure. The primary antibody was first appliedto the tissue sections which were then further labelled with abiotinglated link antibody followed by a streptavidin peroxidase enzymeconjugate. The bound peroxidase enzyme was then visualised with adiaminobenzidine substrate.

[0208] Tumour xenografts of Icc could be imaged with a gamma cameraafter administration of suitable gamma-emitting radionuclide labellingof tumour specific monoclonal antibodies.

[0209] In addition to documentation of tumour cell death and toxicradiation, effects on normal organs, excisional biopsy andhistopathological examination were used for quality control of the modelto ensure the absence of host reaction and rejection of the tumourxenografts.

[0210] Throughout each study, animals were observed for pain anddistress by monitoring food and water intake and observing changes ingeneral well-being and presence of teeth grinding. No post-proceduralpain was anticipated. If changes to these parameters occurred, theanimals were euthanased by intravenous lethabarb. Otherwise, animalswere euthanased within 70 days of tumour implantation.

[0211] Animals were imaged under halothane general anaesthesia whensubcutaneous tumours reached a size of at least 1.0 cm. Gamma imagingwas performed at intervals appropriate to the physical half-life of theradionuclide used to label the monoclonal antibody.

[0212] Results

[0213] Immunesuppression of Sheep with CsA and Ketoconazole

[0214] When the Merino-Dorset lambs used were immunosuppressed, asepticconditions were not required and no sterilization of food and water wasnecessary (in contrast to the sterile environment required formaintenance of nude mice and rats).

[0215] The weight remained relatively constant over the period ofimmunosuppression, as the anorexic effect of CsA was balanced by theanticipated natural weight gain of 3 kg per week.

[0216] Sheep Skin Autografts and Heterografts

[0217] Full thickness sheep skin heterografts were transplanted intoCsA/ketoconazole immunosuppressed sheep, and compared with fullthickness skin autograft transplants. Given maintenance of trough plasmaCsA levels in the optimum range, the skin heterograft appearance wasidentical to that of the adjacent autografts, both macroscopically andmicroscopically. However, if the CsA was stopped or fell below acritical level, rejection processes occurred and the graft becamenon-viable within 10 days.

[0218] Mouse Tumour Xenografts

[0219] Implantation of B16 tumour pieces and inoculation of suspensionof 10⁷ cells at each subcutaneous site gave rise to viable tumours whichregularly attained a diameter of greater than 1 cm at 3 weeks.Inoculations of 10⁷ cells into peritoneum and subserosally in colon,under direct vision at laparotomy, also generated viable B16 melanoticmurine tumour nodules at these intra-abdominal sites in the sheep.

[0220] Human Tumour Xenografts

[0221] The human tumours successfully transplanted into theimmunosuppressed sheep included the human colon carcinoma cell linesHT-29 and LS 174T, which elaborate CEA, and a human amelanotic melanoma,SKMEL and OVCAR-3 and JAM results are represented in FIGS. 14 and 15.

[0222] Subcutaneous inoculations of 10⁷ cells at each site gave rise toviable tumours of 1.5-2 cm diameter within 3 weeks at almost all sitesfor each human tumour type. Histological examination demonstratedabundant mitoses in xenografted tumour cells, with no significantnecrosis and absence of host inflammatory cell reaction. The morphologyof the tumours was true to type in that the less well-differentiatedHT29 colon cancer manifested few glandular structures, and peroxidasestaining demonstrated less elaboration of CEA than thewell-differentiated LS174T tumour, in which abundant CEA activity wasobserved, particularly on the luminal surfaces of the glandularformations. Similarly, mucin production reflected the degree ofdifferentiation of these tumour cells, and was much more prominent inLS174T xenografts.

[0223] Xenografts of SMEL remained amelanotic, and showed abundant S100staining typical of human melanomata.

[0224] Tumour cell spheroids of LS174T cells were also prepared. Thespheroids which were 300 mm in diameter and comprised 8×10³ LS174Tcells, were administered via the portal vein under direct vision atlaparotomy, in an attempt to simulate intrahepatic metastases of humancolon carcinoma in the sheep liver. Similarly, pulmonary metastases weresimulated by the intravenous administration of LS174T spheroids.

[0225] Orthotopic transplantation of LS174T human colon cancer in oursheep was achieved by inoculation of 10⁷ cells into the wall of stomachand colon and hepatic matastases were induced by intravenousadministration by portal vein or simulated by intrahepatic inoculation,and direct sub-peritoneal implantation was also successful. Spontaneousmetastasis to liver or lymph nodes was not observed in these animalspossibly due to the relatively short duration of the experiment (3weeks). Studies of metastasis may be facilitated by orthotopicimplantation of intact human tumours (Fu et al, 1991) which would berelatively easy in the sheep in comparison with mice and may beperformed at multiple sites in the same animal.

[0226] For example, human colon tumour pieces or inoculation of cellsmay be orthotopically implanted submucosally in the distal colon undersigmoidoscopic control without requirement for abdominal surgery. Suchsigmoid tumours may then be monitored endoscopically, and serialbiopsies taken as required. We have used a similar endoscopic approachto orthotopically transplant human bladder carcinoma cells in Matrigelbeneath the vesical urothelium in sheep via an operating paediatriccystoscope. Matrigel, a reconstituted basement membrane matrixpreparation (Fridman et al, 1991), was found to facilitate tumour graftacceptance at sites of cell inoculation, particularly for OVCAR NIH3 andJAM human ovarian carcinoma cells orthotopically transplanted into sheepovaries. Enhancement of tumour growth was also observed followingtransplantation of multicell spheroids of LS174T cells in comparisonwith inoculation of LS174T single cell suspenstion at the same sites. Wealso found that LS174T xenografts grown subcutaneously in nude mice fromcell inoculations, when implanted into the immunesuppressed sheepsubdermally or explanted on the wall of colon as tumour chunks, thexenografts grew more rapidly than xenografts arising from inoculation ofLS174T single cell suspensions. The uptake of ¹³¹I-A5B7 anti-CEAmonoclonal antibody was similar for such implanted tumour pieces to thatobserved in subcutaneous LS174T xenografts originating from inoculationof cell suspensions, and both were demonstrated on gamma camera imagestaken 3-5 days following administration of the radiolabelled anti-CEAantibody.

Example 5 Localization and Imaging of Human Tumour Xenografts UsingRadiolabelled MoAb.

[0227] Intravenous administration of ¹³¹I-radiolabelled anti-CEA MoAb,A5B7 (Celltech Ltd, Slough UK), to CsA/ketoconazole immunosuppressedsheep bearing subcutaneous xenografts of human HT29, LS174T coloncarcinoma and SKMEL human melanoma allowed gamma camera imaging of 1-2cm of colon cancer xenografts at 3 and 5 days after injection of theradiopharmaceutical. The nonspecific control SKMEL tumours were notdetected. The tumour localization of CEA-specific ¹³¹I MoAb wasconfirmed by gamma counting, which showed the greatest accumulation ofradioactivity within LS174T xenografts, a lesser amount in HT29 cellsand only background activity in the nonspecific SKMEL melanomaxenograft.

[0228] The uptake of ¹³¹I-A5B7 in LS174T human colon cancer xenograftsin the immunosuppressed sheep ranged between 0.014 and 0.035% DI/g,higher activities being observed in hepatic sites as shown in Table 6.This tumour uptake is in accord with that achieved in human colonictumours studies in patients using ¹³¹I-A5B7 anti-CEA-monoclonalantibody, where peak uptake of 0.018% DI/g was observed at 27 hoursafter administration of radiolabelled intact antbody (Lane et al, 1994).These modest tumour uptakes in sheep contrast with those achieved inLS174T human colon cancer xenografts in nude mice where ¹³¹I-A5B7 peaktumour uptake is over 20% DI/g (Pedley et al, 1993). These relativelyhigh human tumour uptakes of radiolabelled antibodies are commonlyachieved in nude mouse xenografts (Siler et al, 1993; Senekowitsch etal, 1989) but the typical uptakes for the same antibody and tumour typein man, are around 0.005% DI/g (Dykes et al, 1989; Begent et al, 1990).Expectations of curability of tumours by radioimmunotherapy, based onnude mouse model results are therefore unrealistic. For example, if a 60Gy dose in one week is considered sufficient for tumour sterilizationand given a tumour uptake of ¹³¹I-labelled monoclonal antibody of 0.005%DI/g the corresponding whole body radiation absorbed dose would be 17 Gy(Vaughan et al, 1986). The maximum tolerable whole body dose in man isin fact around 2 Gy, and new approaches to radioimmunotherapy of solidtumours will be necessary. One such approach is regional therapy, andthe comparable size and anatomy of the sheep will facilitate explorationof methods of local and intra-tumoral radioimmunotherapy. For example,we have inoculated our immunosuppressed sheep with human tumour cells inliver, peritoneum and bladder to prvide models for regionalradioimmunotherapy delivered via hepatic artery, or intra-peritoneal andintra-vesical injection. Monitoring by quantitative gamma camera imagingis easily performed in this large animal model and results can becorrelated with counting of biopsy samples and autoradiography tovalidate algorithms for calculation of dosimetry in patients insubsequent clinical trials to evaluate safety and efficacy ofradioimmunotherapy of cancer. TABLE 6 HUMAN TUMOUR XENOGRAFTS INIMMUNE-SUPPRESSED SHEEP ¹³¹I-A5B7 anti-CEA IgG1 Mab % DI/gm: mean (Sd) 1DAY (n = 1) 3 DAYS (n = 2) 5 DAYS (n = 5) 7 DAYS (n = 5) BLOOD 0.0279(0.0039) 0.0263 (0.0008) 0.0142 (0.0059) 0.0124 (0.0028) LIVER 0.0091(0.0014) 0.0066 (0.0001) 0.0051 (0.0032) 0.0040 (0.0018) SPLEEN 0.0027(0.0007) 0.0036 (0.0011) 0.0034 (0.0012) B.M 0.0035 (0.0008) 0.0038(0.0013) 0.0027 KIDNEY 0.0059 (0.0008) 0.0054 (0.0018) 0.0050 (0.0014)HEART 0.0054 (0.0001) 0.0025 (0.0000) LUNG 0.0115 (0.0022) 0.0064(0.0009) SK MEL S/C 0.0040 (0.0011) 0.0027 (0.0014) 0.0040 (0.0020) HT29 S/C 0.0068 (0.0008) 0.0085 (0.0065) 0.0053 (0.0011) LS 174T S/C0.0291 (0.0025) 0.0126 (0.0049) 0.0130 (0.0050) 0.0102 (0.0049) LS 174TSTOMACH 0.0207 0.0266 LS 174T COLON 0.0051 (0.0005) LS 174T PERITONEUM0.0188 0.0208 (0.0052) LS 174T LIVER 0.0342 (0.0068) 0.0111 (0.0036)

[0229] The SKMEL tumours showed typical morphological charcterisitcs ofhuman melanoma and did not accumlate any of the radiolabelled anti-CEAantibody taken up by the colonic xenografts. Only the melanoma wasobserved to metastasize from subcutaneous sites of inoculation and SKMELcells were subsequently recovered from the regional pre-stifle lymphnode, grown in cell culture and reinoculated subcutaneoulsy in sheep andgave rise to tumours morphologically indistinguishable from those of theprimary inouclation. The failure of colon cancer to metastasize fromsubcutaneous sites of inoculation has been observed consistently in nudemice (Fidler, 1990; Kubota, 1994) and orthotopic transplantation hasbeen advocated to maintain the malignant phenotype of human tumourxenografts (Radinsky and Fidler, 1992). For melanoma, the subcutaneousroute of cell inoculation represents orthotopic transplantation andregional lymph node metastases were demonstrated in our sheep.

SUMMARY

[0230] The results of these experiments demonstrate that we haveestablished a large animal model of immunosuppression in the sheep whichallows successful transplantation of skin grafts and tumour xenografts.Tumour cell transplantation in this model can be performed at varioussites (subcutaneous, intra-abdominal, intrahepatic, intrapulmonary andintracardiac) to simulate primary and metastatic tumour localization.Transplantation of tumour spheroids can also be performed.

[0231] The animal model of the invention will enable us to produce bonemarrow metastases of breast cancer by intra-cardiac inoculation oftumour cells, providing a model of bone marrow metastases in breastcancer patients.

[0232] The animal model of the invention will also be useful in thedevelopmental and/or evaluation of novel ligands to permit targetting oftherapeutic agents to the tumours. This may be achieved, for example, byradiolabelling of antibodies raised against these ligands (eg. “tumourspecific” monoclonal antibodies) with therapeutic radionuclides such asRhenium-188, Holmium-166 and Samerium-153 without compromisingimmunoreactivity and without in vivo breakdown of the labelled antibody.

[0233] We have used a similar endoscopic approach to orthotopicallytransplant human bladder carcinoma cells in Matrigel beneath the vesicalurothelium in sheep via an operating paediatric cystoscope.

[0234] Lastly, we have clearly demonstrated the ability to performspecific radioimmuno-scintigraphy of human tumour xenografts in sheep,and this model will aid preclinical evaluation of the efficacy ofpotential radioimmunotherapy for a variety of human metastatic cancers.

[0235] Bladder carcinoma cells BL-17/0/X1; J82; 5637 in Matrigel areinjected into the urothelium of 6-10 sheep via a cystoscope. The sheepare monitored by cystoscopy and small biopsies taken to confirm tumourgrowth and phenotype.

[0236] After an appropriate time, for example 5 weeks, the sheep withvisible tumours are injected intravesically with Samarium-153 labelledC1-137, 595 or cytokeratin-8 or labelled isotype control antibody, orwith labelled EGF or unlabelled EGF as control. Subsequent cystoscopy isperformed 5 and 10 days later to monitor tumour growth. Tumour andsurrounding urothelium are examined histologically at autopsy.

[0237] Yttrium-90 labelled octreotide (Novartis) is a somatostatinanalogue which targets somatostatin receptors on carcinoid, small celllung cancer or the like. This enables radiopeptide receptor therapy tobe studied, using an animal model of the invention, and which has beeninoculated in the liver with human carcinoid BON cells in Matrigel.

[0238] Treatment of glioma by administration of Yttrium-90 octreotidemay also be studied in sheep which have been subjected toorthotransplantation by injection of glioma cells directly into thefrontal lobe of the brain.

[0239] The animal model described herein may also be used for directtransplantation of tumours, including human tumours freshly taken fromsurgical specimens. This has the advantage of preserving the malignantphenotype of the tumour, and the propensity to metastasize whentransplanted orthotopically. The expression of specific tumourassociated antigens, which can be lost during passage in cell culture,can also be preserved.

[0240] The unique attribute of the large animal model of human tumours,in contrast to the mouse, is the ability to measure uptake ofradioactivity in tumours and critical normal organs, by quantitativeSPECT imaging in vivo and to verify the time course of accumulation oftumour radioactivity by serial biopsies for gamma counting. When MoAbsare radiolabelled with isotopes such as Iodine-131, Holmium-166,Samarium-153, Rhenium-186, Rhenium-188, Copper-64, Scandium-47 andLutetium-177, which emit both beta and gamma rays, dosimetry ofradiotherapeutic activities may be measured in vivo and cancericidaleffects verified by serial tumour biopsy and histological andmicroauto-radiographic correlation.

[0241] In addition to documentation of tumour cell death and toxicradiation effects on normal organs, excisional biopsy andhistopathological examination are used for quality control of the modelto ensure the absence of host reaction and rejection of the tumourxenografts.

[0242] Other Uses of the Animal Model for Studying Cancer

[0243] Radioimmunoscintigraphy of metastatic breast carcinoma has beendisappointing (Kahn et al, 1993), but recent clinical testing of a new,commercially available monoclonal antibody BrE-3 which is reactiveagainst a mucin epitope has shown encouraging localization, andtherapeutic potential has been postulated (Kramer et al, 1993). Theanimal model of the present invention is useful to test this hypothesis.

[0244] “Humanized” S193, a murine monoclonal antibody raised againstLewis Y antigen of breast carcinoma, has little cross-reactivity withblood epitopes, and the developmental work currently in progress at theLudwig Institute in New York may result in the availability of¹³¹I-labelled humanised S193 Mab. We are exploring the possibility oftesting this new radioimmunotherapeutic agent in our large animal model.

[0245] Recent reports of enhancement of radioimmunotherapeutic effectson xenografts of human breast cancer in nude mice by using concomitantexternal beam irradiation (Warhoe et al, 1992), and reports ofsynergistic effects of interferon gamma (Buchsbaum et al, 1991) may alsobe investigated in our large animal model.

[0246] In addition to the modelling of human cancer metastases, the CsAimmunesuppressed sheep, having organs of comparable size to those of thehuman, allows direct organ implantation of human tumour cells to createxenografts which simulate primary malignancy. For example, cells fromhuman glioma may be relatively easily inoculated into the brain of theimmunosuppressed sheep to facilitate the study of efficacy of variousprimary treatments such as targeted chemotherapy, radiopharmaceuticaltherapy, or local internal or external radiation treatments.

[0247] Likewise, myeloma cells can be inoculated directly into themarrow of the long bones or spleen in this large animal model.

[0248] The model of the invention may also be used to study the effectsof gene therapy or the control of metastasis by a gene of interest. Forexample, cancer suppressor genes such as the p53 gene, the DCC (deletedin colon carcinoma) gene, the metastasis regulating gene, nm23, or anytumour-suppressor or inhibitor gene of interest which can be insertedinto a vector such as a viral vector or liposome, may be co-implanted orinoculated with cells as described above. Alternatively, geneticallymanipulated cells containing these genes may be transplanted into theanimal model. The genes may also be introduced after xenografting andmetastasis at the site of the tumour formation. The effects of genetherapy alone, or in combination with the forms of therapy alreadydescribed, can then be investigated.

[0249] Whether the goal of treatment is to sterilize primary orsecondary human tumours, the CsA-immune-suppressed sheep model providesan in vivo system of comparable size and physiology to human patientsand allows detailed study of targeted cancer therapy.

[0250] In the particular instance of radioimmunotherapy, the size of theorgans permits tumours which mimic neoplasms in patients presenting withearly cancer to be studied under controlled conditions, particularly byorgan imaging modalities such as gamma camera SPECT, CT or MRI which areimpractical in nude mice or rats.

[0251] In addition to accurate measurement of pharmacokinetics ofpotential therapeutic agents, the tumour specificity and localization inthe human cancer cells can be measured directly, and the timerelationships examined by serial excisional biopsy and histochemical orquantitative microautogradiographic examination, or gamma or betascintillation counting.

[0252] Safe therapeutic application of novel tumour specificradiolabelled monoclonal antibodies requires preclinical delineation ofcritical organ dosimetry such as can be measured by SPECT imaging of alarge animal human tumour model, validated by serial biopsies of majororgans for accurate gamma or beta counting. The target tumour dosimetrycan also be accurately measured by well counting of excisional biopsiesto establish the potential efficacy of any radiopharmaceutical prior toembarking upon a clinical trial. Computer algorithms can then bedeveloped, and tested, to perform prospective critical organ dosimetryon tracer doses of radioimmunotherapeutic agents, validated by directmeasurement in the large animal model as described above, to accuratelyprescribe a maximum safe tolerated dose to a patient before committingto therapy.

[0253] It will be apparent to the person skilled in the art thatalthough the examples have been described in some detail for thepurposes of clarity and understanding, they represent guidelines only.The person skilled in the art will recognise that various modificationsand alterations to the embodiments and methods described herein may bemade without departing from the scope of the inventive concept disclosedin this specification.

[0254] References listed herein are identified on the following pages,and are incorporated herein by this reference.

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The claims defining the invention are as follows:
 1. An animal model ofcancer, comprising a mammal which is immunosuppressed by administrationof cyclosporin and ketoconazole, and which carries a tumour xenograft.2. An animal model according to claim 1, wherein the mammal isimmunosuppressed by increased bioavailability of cyclosporin.
 3. Ananimal model according to claim 1 or claim 2, wherein the ketoconazoleincreases the bioavailability of the cyclosporin to therebyimmunosuppress the mammal.
 4. An animal model according to any one ofclaims 1 to 3, wherein the cyclosporin is selected from the groupconsisting of CsA, CsB, CsC, CsD, CsE, CsF, CsG, CsH, CsI andderivatives, analogues or homologues thereof.
 5. An animal modelaccording to any one of claims 1 to 4, wherein the ketoconazole furtheroptionally comprises one or more compounds selected from the groupconsisting of fluconazole, or derivatives, analogues, homologuesthereof, and calcium channel blockers.
 6. An animal model according toany one of claims 1 to 5, wherein said mammal has a plurality ofxenografted tumours.
 7. An animal model according to any one of claims 1to 6, wherein the tumour is of human origin.
 8. An animal modelaccording to any one of claims 1 to 6, wherein the tumour is ofnon-human origin.
 9. An animal model according to any one of claims 1 to8, wherein the tumour xenograft is obtained by transplantation of tumourinto the mammal, said tumour being selected from the group consisting offresh surgical specimens, tumour cell lines, cells from a solid tumour,spheroids of cancerous cells and tumour pieces obtained from a tumourpassaged in a host animal.
 10. An animal model according to any one ofclaims 1 to 9, wherein the tumour xenograft is from a cancer selectedfrom the group consisting of bladder cancer, ovarian cancer, bowelcancer, colon cancer, lung cancer, breast cancer, brain cancer andmelanoma.
 11. An animal model according to any one of claims 1 to 10,wherein the tumour xenograft is obtained by orthotopic transplantationof the tumour into the mammal.
 12. An animal model according to any oneof claims 7 to 11, wherein the transplantation is performed usingMatrigel as a vehicle.
 13. An animal model according to any one ofclaims 1 to 12, wherein the mammal is a ruminant.
 14. A method ofevaluating the efficacy of a putative therapeutic agent for cancer,comprising the step of administering said agent to an animal accordingto any one of claims 1 to
 13. 15. A method according to claim 14,wherein the putative therapeutic agent is selected from the groupconsisting of cytokines, chemotherapeutic agents, radiopharmaceuticals,internal radioactive agents, and external radioactive agents, andradiolabelled peptides.
 16. A method according to claim 14, wherein theputative therapeutic agent is a gene therapy agent.
 17. A method ofevaluating the efficacy of a method of radioimaging of tumours orneoplasms comprising the step of administering a radiolabelled,tumour-specific antibody to an animal model according to any one ofclaims 1 to
 13. 18. A method of producing a ruminant mammal bearing atumour xenograft, comprising the step of administration of cyclosporinand ketoconazole to the mammal.
 19. A method according to claim 18,wherein the ketoconazole is administered in a drench formulation.
 20. Amethod according to claims 18 or 19, wherein the dose of cyclosporin is2.5 to 3.5 mg per kg administered twice per day and the ketoconazole isadministered twice a day to maintain trough serum levels of cyclosporinwithin the range 750-1500 ng/ml.
 21. A method according to any one ofclaims 18 to 20, wherein the cyclosporin is selected from the groupconsisting of CsA, CsB CsC, CsD, CsE, CsF, CsG, CsH, and CsI.
 22. Amethod according to any one of claims 18 to 21, wherein the ketoconazolefurther optionally comprises one or more compounds selected from thegroup consisting of fluconazole and calcium channel blockers.
 23. Amethod of direct transplantation of a tumour, comprising the step oftransplanting a surgically-removed tumour specimen into a mammal whichis immunosuppressed by administration of cyclosporin and ketoconazole,and optionally allowing the specimen to metastasize in said mammal. 24.A method of stimulating spontaneous metastasis of tumour cells to atarget site, comprising the step of transplanting said cells to a mammalwhich is immunosuppressed by administration of cyclosporin andketoconazole and allowing the cells to metastasize in said mammal.
 25. Amethod according to claim 24, wherein the target site is selected fromthe group consisting of lymph nodes, bladder, ovary, bowel, colon, lung,breast, brain or skin.
 26. A composition comprising CsA or a derivative,analogues or homologues thereof, and ketoconazole or a derivative,analogue or homologue thereof, together with a pharmaceuticallyacceptable carrier.
 27. A kit for establishing an animal model ofcancer, comprising a CsA, a derivative, homologue or analogue thereofand ketoconazole, a derivative, homologue or analogue thereof, whereinthe ketoconazole, derivative, homologue or analogue thereof increasesthe bioavailability of the CsA, derivative, homologue or analoguethereof and enables the establishment of a tumour xenograft.